JPH0258916B2 - - Google Patents

Description

[Detailed description of the invention]

Technical Field of the Invention The present invention relates to Been, Golden Mosaic Virus (hereinafter sometimes abbreviated as "BGMV").
Concerning DNA and hybrid DNA incorporating it. To explain in more detail, the single strand of BGMV
DNA obtained by double-stranding DNA, this DNA
Hybrid DNA obtained by inserting BGMV into a vector for other organisms, Hybrid DNA obtained by propagating this hybrid DNA, and Hybrid DNA obtained by extracting only the BGMV-derived DNA fragment from this hybrid DNA and circularly ligating it. was given
Concerning DNA and replicative DNA of BGMV. Prior Art Recently, DNA genes from viruses have been widely developed and used as vectors in genetic recombination technology. An example of a virus that provides such a vector is the simian virus (SV).
40 and papovaviruses such as polyomavirus,
Papillomaviruses and adenoviruses are known. However, these known vectors were discovered as animal-based vectors, and
It does not proliferate in plant cells. Therefore, these known vectors cannot be used as vectors for plant genetic recombination. To date, vectors that can be used as plant genetic modification vectors include the extranuclear vector possessed by Agrobacterium tumefaciens, which causes tumors in dicotyledonous plants such as tomatoes and tobacco. The only known genes are the Ti plasmid and the DNA gene from the cauliflower mosaic virus, which causes diseases in cabbage and Chinese cabbage.
Other than these, vectors suitable for plant genetic recombination have not yet been developed. Furthermore, it can be said that no plant virus other than the above-mentioned cauliflower mosaic virus is known to have a DNA gene that could potentially serve as a vector for plant genetic recombination. Recently, Robert M. Goodman of the University of Illinois in the United States and his colleagues have discovered that the gene of BSMV, a tropical plant virus, is composed of two paired particles with a diameter of approximately 18 nm. As a result of analyzing its nucleic acid, it was discovered and reported that the gene of this virus is a circular single-stranded DNA with a size of approximately 2500 bases [Virology 83 171 (1977),
See Virology 97 388 (1979)]. Subsequently, several types of plant viruses were discovered that have a form in which two particles with a diameter of about 18 nm pair together to form a single virion, and whose genes are circular single-stranded. are called the “gieminivirus group”. As mentioned above, the only known plant DNA viruses are cauliflower mosaic virus and dieminivirus, and dieminivirus is extremely promising as a target for the development of vectors for plant genetic modification. Conventionally, the Ti plasmid and cauliflower mosaic virus, which have been proposed as vectors for plant genetic modification, can only infect dicotyledonous plants, that is, host plants, whereas dieminivirus can be used not only for dicotyledonous plants, but also for dicotyledonous plants. Monocots such as wheat and corn, which are important grains for humans, can also serve as hosts, so this dieminivirus is
Converting DNA into a vector for plant genetic modification is in itself extremely significant. On the other hand, cauliflower mosaic virus multiplies within the cell chambers of plant cells, whereas dieminivirus multiplies both within the cytoplasm and within the nucleus. This means that when the dieminivirus is made into a vector,
This indicates that it is highly possible to modify the nuclear genes of plants themselves. Therefore, it is clear that it would be industrially valuable if the DNA of dieminivirus could be transformed into a vector. Structure of the present invention Therefore, the present inventors have developed the single-stranded DNA of bean golden mosaic virus (BGMV), which is a type of dieminivirus, into a double-stranded vector that is technically easy to handle in genetic recombination operations, and built-in hybrid
The present invention was achieved as a result of research into DNA and the like. That is, according to the present invention, (i) from BGMV
It is DNA obtained by at least partially making double-stranded single-stranded DNA with a length of about 2.5 kilobases.
DNA showing the restriction enzyme map of (hereinafter referred to as DNA−
(abbreviated as a) and (ii) DNA obtained by at least partially double-stranding a single-stranded DNA with a length of about 2.61 kilobases and showing the restriction enzyme map shown in Figure b.
(hereinafter abbreviated as DNA-b), but according to the present invention, the double-stranded DNA-a and DNA
Hybrid DNA obtained by digesting -b with a restriction enzyme and inserting it into another biological vector cleaved with the same restriction enzyme, and hybrid DNA obtained by returning this hybrid DNA to the original vector host and propagating it. , shows the restriction enzyme cleavage map of diagram a or b obtained by decomposing this hybrid DNA with a restriction enzyme and then ring-closing ligation.
DNA provided. Furthermore, the present invention provides replicative DNA containing the base sequences of these DNAs. Bean golden mosaic virus (BGMV)
is a virus that causes golden mosaic-like disease in kidney beans in Central and South America.
Phytopathology 67 (No. 1) 37 (1977) was used to purify the following single strand from infected plants.
DNA can be isolated and purified. (1) Isolation of single-stranded DNA from BGMV Virus was purified from infected plants by the method described in the above literature, and then the purified virus
500μg in 30mM Tris HCl (PH=7.6) 1%
SDS, Proteene K 10 μg/ml solution 2
Shake for minutes and then perform protein extraction three times with 700μ of phenol. The solution phenol was removed from the aqueous layer by ether extraction, and then the single-stranded DNA of the virus was separated by ethanol precipitation.
Can be purified. (2) Double-stranding the single-stranded DNA of BGMV The single-stranded DNA obtained as described above is then injected into
Double-stranded in vitro. In this double-stranded formation, 0.002 ~
Preferably, it is used at a rate of 2000 μg. As a primer, G.M. Taylor (J.
Biochimica et Biophysica by M.Tayler et al.
An oligonucleotide obtained by degrading calf thymus DNA with DNase can be used by modifying the method described in Acta, 442 325 (1976). The present inventors used primers obtained by this modified method as described in the Examples below. When using this oligonucleotide as a primer, the amount of primer is
50 to 400 μg per μg of DNA, more preferably
A proportion of 100 to 300 μg is suitable. In addition, when using purified DNA containing at least 10 bases and having a completely complementary sequence as a primer to the single-stranded DNA of BGMV, the ratio of primer used is per 1 μg of single-stranded DNA.
The amount may be 0.002 μg to 1 μg, or even 0.004 μg to 0.02 μg. Deoxyadenosine triphosphate (dATP), deoxycytidine triphosphate (dCTP), deoxyguanosine triphosphate (dGTP) and deoxythymidine triphosphate ( dTTP) each at a final concentration of 10-300 μM,
It is preferably added in a range of 50 to 150 μM. At this time, for example, a part of dCTP is converted into d-32P-deoxycytidine triphosphate (d-32P-dCTP).
It is convenient to substitute , as it makes it easy to track the progress of double-stranded DNA and the double-stranded DNA in subsequent reactions. In addition, Mg ⁺⁺ (e.g., magnesium chloride, magnesium sulfate, etc.) is used in a concentration range of 1 to 50 mM, preferably 5 to 21 mM, in order to accelerate the enzymatic reaction. Unless otherwise specified, it means the concentration unit of "mol/".A buffer such as Tris HCl (PH
7.6-8.4) at 30-300mM, preferably 70-300mM
Advantageously, a concentration of 200mM is used. Thus, the above mixture is preferably 50~
Maintain at a temperature of 80°C, especially 60-70°C, for 1-10 minutes, preferably 3-7 minutes, then if necessary
Rapid cooling to ℃ or lower. Note that the total amount of the mixture is based on vector single-stranded
1 to 1000μ, preferably 10 per μg of DNA
It is preferable to set it in the range of ~500μ. It is also effective to use commonly used enzyme stabilizers, such as α-mercaptoethanol and dithiothreitol (DTT). The concentration of the stabilizer in the mixed solution is 0.1 to 5% (by weight), preferably 0.5 to 5% (by weight).
It can be used in a range of 2% (by weight). Next, an enzyme for double-stranding is applied, and examples of the enzyme for double-stranding include Avian myeloblastosis virus (AMV).
reverse transcription factor, T4-DNA polymerase, Escherichia coli DNA polymerase I, DNA polymerase
Examples include large fragment (Klenow enzyme). Among these, AMV reverse transcriptase or DNA polymerase large fragment is preferred. The proportion of double-stranded enzyme used depends primarily on its type. For example, when using AMV reverse transcriptase (code 120248 manufactured by Seikagaku Corporation), it is effective to use 1 to 20 units, preferably 5 to 10 units, per 1 μg of single-stranded DNA. If the number is less than 1 unit, it may be difficult to form double strands sufficiently. After adding the double-stranded enzyme, the reaction is first carried out at a temperature of 10-30°C, preferably 15-25°C for 2-30 minutes, preferably 5-20 minutes, and then 30-45°C, preferably 35-40°C. 30-180 minutes at a temperature of preferably
It is advantageous to react for 50 to 120 minutes. To stop the reaction, add water-saturated phenol or EDTA aqueous solution (PH = 8.0) to the reaction mixture.
You can add a stopper such as In the case of water-saturated phenol, 1/10 to 2
It is only necessary to separate and collect only the aqueous layer by doubling the volume, shaking, and centrifuging, and in the case of an EDTA aqueous solution, it is sufficient to use an amount sufficient to capture Mg ⁺⁺ present in the mixture. The double-stranded DNA may be separated from the DNA-containing aqueous solution that has been subjected to the reaction termination treatment, and a preferred separation operation is a gel filtration method. To explain one specific example of that case, a double-stranded DNA-containing aqueous solution is
Sephadex G-10 [manufactured by Pharmacia Huain Chemical Co., Ltd.] and Biogel p30 [Bio-Rad Co., Ltd.]
The 32p intensity of each fraction is determined by fractionation using a gel filtration reagent such as the one manufactured by J.D. By precipitating the DNA in ethanol or isopropanol, double-stranded DNA can be obtained. The double-stranded DNA thus separated is
Double-stranded DNA based on the single-stranded DNA of BGMV
may contain a considerable amount of primer DNA other than the This tendency is remarkable when oligonucleotides from the calf thymus DNA are used as primers. Therefore, in such cases double-stranded DNA
It is desirable to purify it again to make it even more pure. This purification can be carried out using the gel filtration method described above. To give a specific example, when starting from 20 μg of single-stranded DNA, add the double-stranded DNA to 100 to 500 μg of water.
Dissolved in 10mM Tris BCl (PH=7.4),
Sephadex equilibrated with 100mM NaCl aqueous solution
Gel filtration is performed using a column packed with G-50 or Biogel p30 with enhanced separation ability. 10mM Tris HCl (PH=7.4), 100mM NaCl as eluent
High purity BGMV double-stranded DNA can be obtained by collecting the fraction with a high 32p count from the void volume using a method and precipitating it in ethanol or isopropanol. (3) Double-stranded DNA There are two types of DNA in the dieminivirus in BGMV described above, and there are also two types of DNA obtained by double-stranded DNA as described above. This 2
The double-stranded DNA of a species can be separated into individual DNAs, or can be cloned and then separated. Also, before making double strands (in other words, in the state of single stranded DNA), separate each
Double stranding can also be performed in the same way for each. DNA-a mixture of single-stranded DNA of BGMV
To separate DNA-b and DNA-b, a method called “strand separation” is used [for example, in the book “Molecular
Cloning A Laboratory Manual” No. 180~
[method described on page 185] That is, a method using gel electrophoresis, or a DNA fragment complementary to either DNA a or b is added to a cesium chloride solution of a DNA mixture, and single strands are isolated by equilibrium density gradient centrifugation. It is possible to employ a separation method that utilizes the density difference between the DNA that remains intact and the partially double-stranded DNA. However, it is easier to separate DNA-a and DNA-b by converting the single-stranded DNA mixture into double-strands, for example, cloning them into plasmid pBR322, etc., and then separating them. This separation method will be explained in detail later. Thus, the DNA obtained by double-stranding the DNA from BGMV has a size of 2.57 ± 0.1 kilobase pairs (hereinafter "kilo base pair" may be abbreviated as "Kpb"). The restriction enzyme cleavage map of Figure a is shown. The other one has a size of 2.61±0.1 kilobase pairs (Kbp) and shows the restriction enzyme cleavage map shown in Figure b. In Figure a, each restriction position is shown as a positional coordinate with the Hind cleavage site as [0.0/2.57], and the unit is kilobase pair (Kbp). In addition, in Figure b, each restriction site is shown as a position coordinate, with one position of Hind being [0.0/2.61]. Next, the fragments generated by digestion with each restriction enzyme are shown in Table 1 below. In the table, the fragments that appeared densely compared to the size of the fragments were divided into Group A and the others were classified into Group B.

[Table] In the table above, (3.3) means that the DNA remains as an open ring without being cut at any point, and the apparent molecular weight is 3.3 Kbp relative to the size marker of linear DNA. It shows. It should be understood that the values for the size of fragment DNA in the table above may vary by ±0.1 Kbp, especially ±0.05 Kbp due to the accuracy of experimental analysis. For restriction enzyme digestion, use a concentration of 10mM Tris HCl.
(PH=7.9), 7mM MgCl ₂ , 7mM β-mercaptoethanol, and 0.01% bovine serum albumin were used as the base solution. Decomposition with Cla is performed with the above basic solution, Hind, Ava, Bgl, Kpn, Pvu
For the decomposition, NaCl was added to the base solution to a concentration of 50mM, and Sal, BamH, Xba,
For Xho decomposition, add 150mM NaCl to the base solution.
I added it so that In the present invention, restriction enzymes include Hind, Sal,
Bgl, BamH, Kpn, Pst, Pvu and
For Xho, we use products manufactured by Takara Shuzo Co., Ltd., for Cla, we use products manufactured by Boehringer Mannheim, and for Ava and Xba, we use products manufactured by Bethesda Research Laboratories. did. In the present invention, all control enzymes for DNA cleavage were used at a ratio of at least 4 units/1 μg of DNA, and the digestion was carried out at 37° C. for 4 hours or more. When using two types of restriction enzymes, first digest with a low salt concentration enzyme at 37℃ for 2 hours or more, then adjust to a high salt concentration, and further with a high salt concentration enzyme.
Decomposed at 37°C for over 2 hours. Thus, the DNA after enzymatic degradation was analyzed by electrophoresis on a 1.5% agarose gel containing 0.5 μg/ml of ethidium bromide to identify the fragments generated by each enzymatic degradation.
We conducted DNA analysis. In addition, during this electrophoresis,
EcoR of λ-DNA as a DNA size marker
/Hind digested product and plasmid
pBR322 was digested with Tap and used. The double-stranded DNA according to the present invention only needs to be at least partially double-stranded compared to the single-stranded DNA, and is not necessarily double-stranded over the entire region. Not necessary. Since the double-stranded DNA of the present invention is used, for example, as a vector for plant genetic recombination, the DNA is used after being cut with a certain enzyme. In that case, as long as the site to be cut is at least double-stranded, the non-double-stranded site will be repaired and almost completely double-stranded by subsequent cloning or propagation. Therefore, the double-stranded DNA of the present invention only needs to be at least partially double-stranded, but desirably at least 80% of the base pairs of single-stranded DNA, preferably at least
It is sufficient that 90% of the chain is double-stranded. a and b
Different values of the DNA ratio can be obtained depending on the growth process of BGMV, but generally the a/b (mole) ratio is 3 or more. Therefore, as shown in Table 1 above, A
Grouping is possible into group fragments and B group fragments. Double-stranded DNA-a and b of the present invention can be used alone or in combination as a vector for plant genetic recombination. Also
DNA a or b can be cleaved at a specific restriction site with a restriction enzyme to form linear DNA.
A third cut of this DNA using the same restriction enzyme
It can be mixed with DNA in approximately the same molar ratio and linked using a DNA binding enzyme (e.g. T4-DNA ligase) using a conventionally known method to form a circular DNA containing a third DNA at a specific site of a or b. . For example, if the third DNA encodes a gene that confers resistance to the antibiotic Kanamaishi, this third DNA
By using DNA that incorporates DNA,
Plant cells can be transformed to become kanamycin resistant. In this case, transformation methods include, for example, a method in which the above-mentioned DNA is applied to protoplast-formed plant cells in the presence of Ca ⁺⁺ , polyethylene glycol, or in the presence of Ca ⁺⁺ in a high pH state; There is a method of mechanically introducing the above-mentioned DNA into a cell (microinjection). Moreover, the double-stranded DNA a or b of the present invention,
Preferably an enzyme capable of cleaving at one site, e.g.
It can be digested with Hind, Cla or Bgl and then inserted into other biological vectors cleaved with the same restriction enzymes as shown below. In this case, other biological vectors include conventionally known vectors such as plasmid vectors for E. coli (e.g. pBR322),
cosmid vectors (e.g. pKY2662), phage vectors (e.g. Syaron 10), plasmid vectors for Bacillus subtilis (e.g. pUB110, pSA0501),
Vectors for yeast (eg, YRp7) can be used. Hybrids with these other biological vectors
The method for preparing DNA consists of double-stranded DNA-
A mixture of a and DNA-b is completely digested using, for example, Hind, and this is mixed with a Hind-digested product of other biological vectors, such as plasmid pBR322 for E. coli, and treated with a DNA-binding enzyme (e.g., T4-DNA ligase). By cyclizing the bond with
Hybrid DNA of aDNA and pBR322 can be obtained. Similarly, a mixture of double-stranded a-DNA and b-DNA is completely degraded with Cla, and this is mixed with the Cla-digested product of pBR322,
Hybrid DNA of b-DNA and pBR322 can be obtained by cyclization using a DNA binding enzyme. When obtaining these hybrid DNAs, it is better to mix the two DNAs you want to combine in approximately equimolar proportions to more efficiently produce the desired hybrid DNA.
DNA can be obtained. In addition, for other biological vectors, it is recommended to dephosphorylate the 5′ end of the DNA by treating with alkaline phosphatase after cleavage with restriction enzymes.
This is preferable because when preparing DNA, self-circularization can be largely prevented by using vectors for other organisms alone. The hybrid DNA thus obtained is also at least partially double-stranded, like the DNA in Figure a or Figure b, and can be used as a vector for plant genetic recombination. This hybrid DNA is transferred to the original host of other biological vectors, such as E. coli in the case of a hybrid DNA with a vector for E. coli, and Bacillus subtilis in the case of a hybrid DNA with a vector for Bacillus subtilis.
Furthermore, hybrid DNA with yeast vector
In the case of yeast, for example, Escherichia coli and Bacillus subtilis are transformed into competent cells according to known methods, and in the case of yeast, they are treated with Zymolys to form spheroplasts. Transformants can be transformed and selected using appropriate drug resistance or auxotrophy, or by colony hybridization. To explain in more detail one specific example of this method, for example, hybrid DNA is Hind in Figure a.
If the hybrid DNA is a hybrid DNA obtained by ligating a degraded product (Cla degraded product in the case of b) with a Hind/digested product of E. coli plasmid pBR322 (Cla degraded product in the case of b) using T4-DNA ligase, this hybrid DNA is Mandel and Higa's method [J.
Mol. Biol. 53 159 (1970)]
HB101 was transformed and colonies generated on agar (L-Agar) plates containing 50 μg/ml ampicillin were subjected to colony hybridization using the method of Grunstein and Hovnes [Proc. Natl. Acad. Sci. 72 3961]. (At this time, 32p-labeled BGMV DNA is used as a probe).
Select colonies with DNA that hybridizes with the DNA. In this way, figure a (or b)
A hybrid in which DNA and plasmid pBR322 are joined at Hind (Cla in case of b)
Escherichia coli HB101 transformed with DNA can be obtained. Hybrid DNA can be isolated from the transformant thus obtained by a known method. The isolated hybrid DNA is almost completely double-stranded throughout, and it is possible to transform plant cells by inserting a third DNA into a specific site, and it can also be grown in bacteria such as Escherichia coli. It is possible to do so. In order to prepare a large amount of the desired hybrid DNA, a bacterial strain containing the hybrid DNA is grown, and if necessary, operations are performed to amplify only the hybrid DNA to lyse the bacterial cells, and the DNA is extracted from the aqueous solution containing the hybrid DNA. Just separate it. When the hybrid DNA is, for example, a hybrid DNA with the plasmid pBR322 for E. coli, an E. coli transformant containing the hybrid DNA is used, for example, in the experimental book "Molecular Cloning-A Laboratory" by Maniatis et al.
Manual”〔Cold, psring Harbor Laboratory
(1982)] using the methods described on pages 88 to 94 to obtain DNA using amplification of hybrid DNA and various lysis methods, and using a covalently closed cycle (hereinafter referred to as
This makes it possible to separate only hybrid DNA of the type (abbreviated as “ccc”). In addition, the hybrid isolated in this way
Figure a (or b) can be obtained by digesting the DNA with the same restriction enzyme used for cloning.
It can be divided into DNA fragments and vector DNA fragments. Preferably, the DNA fragment shown in Figure a (or b) is separated and then ligated and circularized using T4-DNA ligase or the like. To separate only the DNA fragment shown in figure a (or b), generally agarose gel electrophoresis is performed, and the band portion of the DNA fragment shown in a (or b) is cut out from the gel, as described on pages 164 to 172 of the experimental book from Maniatis mentioned above. According to various methods of
DNA fragments can be separated and purified using gel. The DNA fragments thus separated are combined and circularized using a known method, for example, using T4-DNA ligase. A part of the circularized substance is formed. This single self-circularized substance becomes an apparent 3.3 Kbp band in agarose gel electrophoresis. DNA is extracted and purified from this banded gel in the same manner as described above. This DNA is completely double-stranded throughout the ring and is useful as a vector for plant genetic recombination. Next, the replicative DNA in the present invention will be explained. When BGMV infects and proliferates, The replication type of BGMV is detected from the infected leaves of infected leaves, such as top crop beans (for example, top crop of Taiki Seed Co., Ltd.).
To isolate DNA, use the method of WDO Hamilton et al. [Refer to Nucl. Acid 5 Res. 10 4902 (1982)] or the entire method described in "Introduction to Plant Cell Breeding (Gakkai Publishing Center)" by Atsushi Hirai et al., pp. 86-88. This can be done using a DNA extraction method. Total DNA extracted according to these methods was subjected to 0.8% agarose gel electrophoresis (at this time, λ was used as a DNA size marker).
-EcoR/Hind degradation products of DNA are run together), and then the DNA is transferred from this gel to a nitrocellulose filter (see E.M. Southern, J.Mol.Biol 98, 503-517 (1975)).
of heat-denatured BGMV transferred and labeled with 32p.
Using DNA as a probe, Maniaiatis et al.'s ``Molecular Cloning-A Laboratory''
DNA-DNA hybridization is carried out in the same manner as described on pages 387 to 389 of the same book, and then autoradiography is carried out in the same manner as described on pages 470 to 471 of the same book. The DNA portion that hybridizes with the 32p-labeled BGMV DNA appears as a black band on the X-ray film during autoradiography, indicating that this portion is BGMV-derived DNA. This band is approx.
Ten types are recognized, and their apparent sizes are >20Kbp, 9.5Kbp, 6.8Kbp, 5.2Kbp,
3.8Kbp, 3.1Kbp, 2.5Kbp, 1.9Kbp, 1.5Kbp,
It is 0.87Kbp. DNA that gives these bands
can be present in various forms [e.g. CCC, oven circle (OC), linear monomers and dimers of various forms,
It is inferred that the band is composed of many different types of bands, such as trimers. Among these bands, the 0.87Kbp band is the single-stranded DNA of BGMV.
, but all others are double-stranded DNA.
This is the replicative DNA of BGMV. These replicative intermediate DNA bands are removed from the agarose gel, and only the DNA is separated from the gel using the same procedure as above, and when digested with Cla, Hind, and Sal, the same DNA as in Figures a and b is obtained. You can get fragments.
By doing this, 9 other than DNA equivalent to 0.87Kbp
The DNA that gives each type of band is shown in figure a or b.
It takes various forms based on DNA.
This is the replicative intermediate DNA of BGMV. A portion of the DNA that gives these bands, equivalent to 1.5 Kbp,
DNA equivalent to 3.1Kbp, DNA equivalent to 9.8Kbp
Since DNA is present in relatively large amounts, it is easy to use. Thus, the equivalent of 1.5Kbp, 3.1Kbp or 9.8Kbp
By digesting the DNA with Hind and cloning it into the Hind site of the E. coli plasmid pBR322 using the method described above, a hybrid DNA containing the entire a-DNA can be obtained as described above. In addition, 1.5Kbp (or 3.1Kbp or 9.8Kbp) of DNA was
By degrading the b-DNA and cloning it into the Cla site of pBR322, a hybrid DNA containing the entire b-DNA can be obtained as described above. When DNA obtained by cloning is replicated in E. coli that has a dam methylase gene, certain base sequences, for example, the N at position 6 of A in GATC, are methylated, and in the DNA obtained by cloning, the restriction enzyme of the original DNA is methylated. Although it may be observed that the site appears to have disappeared, the base sequence itself is the same. Furthermore, DNA obtained through cloning has the same infectivity as a virus, just like DNA extracted directly from a virus. The present invention will be described in detail below with reference to Examples, but the present invention is not limited thereto. Example 1 a Virus isolation and purification 57gr of BGMV-infected top crop leaves were collected at 250g.
ml of 0.1M sodium phosphate - 10mM MEDTA buffer (PH=7.8) (contains 1.3gr of cysteine)
Grind the mixture, filter it through double gauze, and drain the liquid.
Centrifugation was performed at 10,000G for 40 minutes at 4°C to obtain 205ml of supernatant. To this supernatant, add 2.4g of salt, then 8.2g of polyethylene glycol (7800-9000), stir at 4℃ for 1 hour, and then apply 10000G for 25 minutes at 4℃.
It was centrifuged at After removing the supernatant, 10 ml of 0.1 M sodium phosphate buffer (PH = 7.8) was added to the precipitated polyethylene glycol pellet to homogenize it, and this was centrifuged at 10,000 G for 30 minutes at 4°C.
The supernatant was removed, and the supernatant was centrifuged at 4° C. for 4 hours at 30,000 rpm using an ultracentrifuge SW40.1 rotor manufactured by Beckman to obtain a crude virus as a precipitate.
After homogenizing this crude virus in 0.5 ml of 0.1 M sodium phosphate buffer (PH = 7.8),
−40% continuous sucrose density gradient centrifugation (same as above)
Using SW40.1 rotor, 32,000 rpm 3 hours 4℃)
After centrifugation, extract 0.6ml from the bottom of the centrifuge tube.
Fractions were collected one by one. The absorbance _A260 of each fraction was measured, and virus peaks were observed in fractions No. 9 to 16. These fractions were collected and homogenized by adding twice the amount of 0.1M sodium phosphate buffer (PH = 7.8). , 35,000 rpm 3 hours 4 with Sw40.1 rotor
A virus pellet was obtained at °C, homogenized in 0.1M sodium phosphate buffer (PH = 7.8), and again subjected to 10-40% sucrose continuous density gradient centrifugation (29,000 rpm 3 hours at 4 °C Sw40.1 rotor). A fraction of 0.6 ml was collected from the bottom of the centrifuge tube.
Only virus peaks were obtained in fractions No. 13 to 18. These fractions were collected, and after homogenization by adding twice the amount of 0.1M sodium phosphate buffer (PH = 7.8), they were heated at 36,000 rpm for 3 and a half hours at 4°C Sw40. The virus was precipitated using one rotor to obtain purified BGMV. b Isolation of single-stranded DNA genes from viruses
Purification Add 750μ of sterile water and 15μ of the purified BGMV above.
1MTrisHCl (PH7.6), 75μ 10% Sodium Dodecyl Sulfate (SDS) 7.5μ Proteinase K
(1μg/μ solution) was added and shaken at room temperature for 2 minutes, then 700μ of phenol saturated with 10mM TrisHCl (PH7.6)-1mM EDTA aqueous solution was added, shaken for 3 minutes, and centrifuged for 5 minutes in an Etzpendorf small centrifuge to remove the aqueous layer. I took it out. This aqueous layer was subjected to the same phenol extraction procedure twice more to obtain a final aqueous layer of 950μ, and then the aqueous layer was extracted with chloroform.
After adding 700μ of ether and shaking for 2 minutes, remove the aqueous layer and add 700μ of ether to extract the phenol. This extraction operation was performed three times. Add 100μ of 3M sodium acetate buffer (PH4.8) and 2.5ml of ethanol to 1000μ of the resulting aqueous layer.
was added and kept at -20°C overnight, and centrifuged for 20 minutes at 35,000 rpm in a Beckman ultracentrifuge with a Sw50.1 rotor to precipitate DNA. This DNA
It was dissolved in 600μ of an aqueous solution of 10mM TrisHCl and 0.1mM EDTA to obtain a 0.5μg/μ aqueous solution of BGMV single-stranded DNA. c In vitro duplexing of BGMV single-stranded DNA; 2μ of BGMV single-stranded DNA (0.5μg/μ), 155μ of sterile water, 30μ of 1M TrisHCl (PH=8.0),
Mix 12μ of calf thymus DNA oligonucleotide (16.6μg/μ) prepared by the method below, hold at 70℃ for 3 minutes, then rapidly cool to 0℃,
30μ _of 80mM MgCl, while keeping at °C.
10% β-mercaptoethanol aqueous solution 30μ,
0.8mM deoxyadenosine triphosphate, 0.8mM
30μ each of deoxyguanosine triphosphate and 0.8mM deoxythymidine triphosphate;
0.8mM deoxycytidine triphosphate 6 μ and deoxycytidine 5′-[α-32p] triphosphate (approximately 3000 Ci/mmol manufactured by Amashiyam Japan Co., Ltd.,
10mCi/ml code number pB10205) 2.4μ and
AMV reverse transcriptase (Seikagaku code 120248,
After adding 2μ of phenol (5 units/μ) and reacting for 10 minutes at 20℃ and 1.5 hours at 37℃, 50μ of phenol was added.
After shaking, the mixture was centrifuged for 5 minutes using an Etzpendorf centrifuge, and the aqueous layer was filtered through a gel. Gel filter
Sephadex G-75 (manufactured by Pharmacia Fine Chemical Co., Ltd.) equilibrated in advance with a 10mM TrisHCl (PH7.4)-0.1M NaCl aqueous solution was passed through a column packed in a 6mm diameter tube with 5ml. After adding the reaction solution, separate 4 drops (approximately 700μ) and separate each fraction.
32p intensity was measured. The first peak appears in fractions No. 5 to 12, and unreacted α-32p-
A peak due to deoxycytidine triphosphate was observed. Collect fractions No. 5 to 12 and add 60μ of 3M NaCl.
Add 1.8ml of ethanol and hold at -20℃ for 3 hours.
25000rpm for 20 minutes (Betskuman ultracentrifuge Sw50.1
rotor), centrifuge, remove the supernatant, and store at -20°C.
Add 2 ml of cooled 70% ethanol aqueous solution to 20,000 ml.
The mixture was centrifuged at rpm for 2 minutes and the supernatant was removed. Precipitate DNA 20
Dry under reduced pressure of mmHg for 1.5 minutes. The obtained DNA
Dissolved in 100μ of 1mM TrisHCl (PH = 7.4) - 0.1mM EDTA aqueous solution and double-stranded in vitro.
Get DNA. (The intensity of 32p was 46×10 ⁴ cpm) Preparation of calf thymus DNA oligonucleotide 66 mg of calf thymus DNA was added to 0.1M NaCl 10mM.
_MgCl2 , dissolved in 6.6ml of 10mM TrisHCl (PH7.4),
460 μg of DNase (manufactured by Millipore Corporation (2182 units/g)) was added, incubated at 37° C. for 3 hours, and 0.66 ml of 0.2 MEDTA was added to react. This reaction solution was subjected to protein extraction three times with 7 ml of water-saturated phenol, and then extracted three times with 7 ml of ether to remove phenol from the aqueous layer. Add 20% ethanol to the resulting aqueous layer.
ml was added, left at -20°C for 2 hours, and centrifuged at 4000G for 4 minutes to obtain a DNA pellet. This DNA pellet
Sephadex G-75 (manufactured by Pharmacia) dissolved in 0.5 ml of 0.1M NaCl 10mM TrisHCl (PH7.4) and pre-equilibrated with 0.1M NaCl 10mM TirsHCl (PH7.4)
(column length: 42 cm, column diameter: 0.5 cm) and aliquots of approximately 1.2 ml were collected. A large peak was observed in the crackles No. 10 to 32, and the fractions No. 16 to 26 were collected (total volume 12.5 ml), 31 ml of ethanol was added, left at -20°C for 30 minutes, and then centrifuged (4000G for 8 minutes). The resulting DNA pellet was dissolved in 1 ml of distilled water (DNA concentration 16.6 mg/ml) and used as primer DNA. Comparative example 1 Calf thymus DNA oligonucleotide (16.6μg/
μ) is 1.5μ instead of 12μ, otherwise it is 1c
When a double-stranded experiment was conducted under exactly the same conditions as the above experiment, the count of 32p in the polymeric DNA part was 8.
×10 ⁴ cpm. It can be seen that less than 1/5 of the double stranding has occurred compared to 1c. Comparative Example 2 The primer was prepared by G.M. Theiler et al.
Biochimica et Biophysica Acta 442 325
Primer mixture from calf thymus DNA prepared by the method of (1976) (DNA concentration 5 μg/μ)
40μ, the oligonucleotide for the 1c experiment (16.6μ
g/μ) Used instead of 12μ, sterile water 155μ
Double stranding was performed in exactly the same manner as in the 1c experiment, except that 127μ was used instead of 1c. polymer DNA
The 32p intensity of the part was 15×10 ⁴ cpm. The double-stranded DNA obtained in Comparative Example 2 was
restriction enzyme Cla under exactly the same conditions as in experiment 1d.
When the DNA was digested with Hind, Sal, Bgl, etc. and then subjected to autoradiography in exactly the same manner as in experiment 1d below, the DNA of the Hind-digested DNA had not moved from the slot position of the agarose gel. In addition, some of the DNA digested with other restriction enzymes remained in the slot position, and the DNA that migrated through the gel also became quite smeared, and the bands were not as beautiful as in the case of 1d. d Degradation and disassembly of double-stranded DNA using restriction enzymes, restriction enzyme cleavage map 32p-labeled double-stranded DNA (32p
Strength 46× ¹⁰⁴ cpm/100μ) 3μ (32p strength
7300 cpm) and buffer for restriction enzyme digestion [100mM TrisHCl (PH7.9), 70mM MgCl ₂ ,
For Cla decomposition, use 70mM β-mercaptoethanol and 0.1% bovine serum albumin as the base solution.This base solution is called the buffer for restriction enzyme digestion.
Ava, Hind, Bgl, Kpn, Pst,
In Pvu decomposition, NaCl is added to the above basic solution.
The solution added to 500mM is called restriction enzyme digestion buffer, and Sal, BamH
, Xba, and Xho decomposition, the basic solution is further
A solution containing 1500mM NaCl is called a restriction enzyme digestion buffer] 4μ, distilled water 32μ, and the restriction enzymes Ava (2 units/μ) and BamH (6 units/μ) shown in Table 1.
Bgl (6 units/μ), Pat (5 units/μ), Xba (6 units/μ),
Hind (5 units/μ), Kpn (6 units/μ), Pvu (6 units/μ),
Sal (6 units/μ), Xho (7.5 units/μ), Cla (5 units/μ),
[Among the restriction enzymes in Table 1, Cla is Ava manufactured by Boehringer Mannheim, Xba is manufactured by Bethenuda Research Laboratory, and the others are Takara Shuzo Co., Ltd.
Made in Japan. ]4 units added and heated at 37℃ for over 4 hours
DNA was degraded. When decomposing with two types of restriction enzymes, first add a low-salt concentration enzyme, hydrolyze for 4 hours at 37℃ at a salt concentration suitable for the enzyme, and then add a salt suitable for a high-salt concentration enzyme. After adjusting the concentration, an enzyme for high salt concentration was added, and further hydrolysis was carried out at 37°C for 4 hours. After hydrolysis, 8μ of a solution of 0.25% bromophenol blue, 50% glycerol, and 10% SDS was added, heat treated at 65°C for 5 minutes, and then 1.5% agarose gel electrophoresis was performed. The agarose used was for type electrophoresis manufactured by Sigma. A 40mM Tris acetate, 2mM MEDTA (PH8.0) aqueous solution was used as an electrophoresis buffer.
At a voltage of 1.5 V/cm in a horizontal gel with a thickness of 5 mm
Electrophoresis was performed for 11-15 hours. During this electrophoresis, λ-
0.5 μg of DNA was completely digested with EcoR and Hind, and 0.2 μg of pBR322 DNA was completely digested with Tap. After electrophoresis,
The agarose gel was taken out, and after drying on a gel drying plate, autoradiography was performed as described in pages 470 to 472 of the experimental book by Maniathes et al. described in the main text. As a result, the sizes of DNA fragments degraded and observed with various restriction enzymes are shown in Table 1 of the text. Furthermore, after autoradiography, it was observed that
The DNA fragments were classified into Group A, which is a group of fragments that appear more densely compared to the size of the fragment, and Group B, which is a group of fragments that appear lighter compared to the size of the fragment. Furthermore, 3.3 in parentheses indicates that the DNA is in an open circle (OC) state and the apparent molecular weight is 3.3 Kbp relative to the linear DNA size marker. From this table, it is thought that the DNA that produces the fragments of group A is open circle (OC) shaped and approximately 2.57 Kbp in size before being digested with restriction enzymes. The exact size can ultimately be determined by determining the base sequence, but with the current method, a minimum measurement error of 0.05 Kbp is unavoidable. The size of the DNA that produces group A fragments can be described as approximately 2.57±0.1 Kbp.From the same consideration, the DNA that produces group B fragments is OC-shaped and has a size of (2.61±0.1) Kbp before being digested with restriction enzymes. be written. Next, by analyzing the degradation patterns of various restriction enzymes alone and in combination, the relative positional relationships of the various restriction enzyme cleavage points are determined, and the intact DNA that produces Group A DNA fragments is determined. Figure a in the text is obtained as a restriction enzyme cleavage map for DNA of group B.
Figure b in the text is obtained as a restriction enzyme cleavage map for intact DNA. e Hybrid DNA of Hind-digested double-stranded DNA and plasmid pBR322 for E. coli; 100μ of double-stranded DNA obtained in Example c
50μ of the solution was pre-mixed with 10mM TrisHCl (PH
7.4), Biogel equilibrated with 100mM NaCl aqueous solution
Gel filtration was performed using a column packed with P30 to a diameter of 6 mm and a length of 30 cm. as eluent
Using 10mM TrisHCl (PH7.4) and 100mM NaCl aqueous solution, 4 drops (approximately 400μ) were collected. 17th
A high count peak of 32p was seen in the ~21 fraction, and when the optical density OD ₂₆₀ of the fraction was measured, the 28th
The OD value started to increase from the fractions and showed large values in the subsequent fractions. Collect fractions 17 to 21 (double-stranded DNA) (2.1 ml), add 200μ of 3M sodium acetate aqueous solution (PH4.8) and 4 ml of ethanol, hold at -20°C for 2 hours, and then centrifuge in a Beckman ultracentrifuge. Centrifugation was performed at 30,000 rpm for 30 minutes in a Sw50.1 rotor. The supernatant was discarded, 5 ml of 75% ethanol at -20°C was added, and the mixture was centrifuged at 20,000 rpm for 2 minutes, and the supernatant was discarded again to obtain a DNA pellet. 2mmHg2
After vacuum drying for a minute, the DNA pellet was washed with distilled water.
To 50μ of this DNA solution, 6μ of the aforementioned restriction enzyme Hind digestion buffer (described in Example C) and 3μ of restriction enzyme Hind were added and reacted at 37°C for 4 hours. Then 10μ yeast
A tRNA (2 μg/μ) solution and 70 μl of distilled water were added, and 100 μl of water-saturated phenol was added, followed by shaking and centrifugation to remove only the aqueous layer. From this aqueous layer, ether extraction was performed three times to remove phenol. Add 10μ of 3M sodium acetate (PH=4.8) and 350μ of ethanol, let stand at -20℃ for 4 hours, centrifuge at high speed, and dry the resulting DNA pellet.
of distilled water. For this DNA 17μ solution, as shown below:
1μ of pBR322 Hind/alkaline phosphatase treatment (DNA 1μg/μ) and DNA binding enzyme buffer [650mMTrisHCl (PH=
7.4), 65mMMgCl, 10mMDTT, 4mMATP,
Add 2μ of 40mM MSpermide aqueous solution and 0.5μ of T4 DNA ligase (manufactured by Takara Shuzo Co., Ltd. code No. 2010B1.2 units/μ) and react at 14°C for 22 hours.
Next, treat the hybrid DNA at 65℃ for 5 minutes.
get. Next, we used this hybrid DNA to infect E. coli.
Transform HB101 compatible cells. pBR322 BamH/alkaline phosphatase treatment solution; pBR322 Hind/alkaline phosphatase treatment (DNA 1μg/μ) solution plasmid
Add the above to a 340μ aqueous solution containing 50μg of pBR322DNA.
Hind restriction enzyme buffer 40μ and Hind
Add 20μ and react for 10 hours at 37℃, then add 1MTrisHCl (PH
8.0) was added, 10μ of bacterial alkaline phosphatase (BAP) (0.4 units/μ, manufactured by Worthington) was added, and the mixture was treated at 65°C for 7 hours. This reaction dephosphorylates the 5' end of DNA.
This reaction solution was subjected to protein removal using 400μ of water-saturated phenol. Next, protein was further removed twice with 400μ of a phenol/chloroform (4/1 volume ratio) mixture, and finally, the phenol component was extracted from the aqueous solution three times with 600μ of ether. 30μ of 3M sodium acetate and 110μ of ethanol were added to the 350μ of the aqueous layer, allowed to stand at −20°C for 2 hours, and centrifuged at high speed. The resulting DNApellet was dried and dissolved in 50μ of distilled water. This is used as the pBR322 Hind/alkaline phosphatase treatment (DNA 1 μg/μ) solution. Preparation of competent cells of E. coli HB101 and their transformation; A single colony of E. coli HB101 was grown in culture medium L.
The cells were transferred to 5 ml of -broth and cultured with shaking at 37°C for 11 hours. This 2 ml was inoculated into 200 ml of new L-broth, and cultured with shaking at 37°C for 2 hours and 20 minutes. When the OD ₆₀₀ reached 0.40,
Cool the culture solution to 0°C and use Tommy's refrigerated high-speed centrifuge.
Centrifugation was performed at 5000 rpm for 5 minutes in a No. 9 rotor. Discard the supernatant, homogeneously disperse the precipitated E. coli in 50 ml of 10 mM NaCl water, centrifuge again at high speed (No. 4 rotor, 5000 rpm for 5 minutes) to precipitate the bacteria, and add 60 ml of 30 mM CaCl ₂ water to the bacterial pellet to homogenize the whole. The temperature was maintained at 0°C for 20 minutes. High-speed centrifugation again (No. 4 rotor, 4000 rpm for 5 minutes)
Discard the supernatant and add _10ml of 30mMCaCl2 to the bacterial pellet.
Add a 15% aqueous glycerol solution to gently homogenize the whole, dispense 200μ aliquots into 1.5ml Eppendorf tubes, and store frozen at -80°C. this
Escherichia coli HB101 treated with CaCl ₂ (referred to as competent cells) was transformed. For transformation, return the competent cells to 0°C, and after about 10 minutes,
Perform the binding reaction with T4 DNA ligase and then 65℃5
The hybrid DNA aqueous solution treated for 1 minute was added, held at 0°C for 40 minutes, and then heated at 42°C for 2 minutes. Next, 1.5 ml of L-broth was added and kept at 37°C for 1 hour, and the culture solution was spread on 8 L-agar plates (diameter 9 cm) containing 50 μ/ml ampicillin at a rate of about 200 μ per plate. Hold at 37℃ for 16 hours
41 transformed HB101 colonies were obtained.
These 41 colonies were plasmidized using the Boiling Lysis method described in the experimental book by Maniathes et al., page 368.
Perform a mini-prep of DNA, then plasmid DNA
Hind disassembled. As a result, 2.58Kbp, 1.62Kbp,
A plasmid containing 1.02Kbp was obtained. 2.58 Kbp was obtained by Hind cleavage of the DNA shown in Figure a, and 1.62 Kbp and 1.02 Kbp were two types of fragments generated by Hind cleavage of the DNA shown in Figure b. 2.58Kbp fragment and
pBGH1, hybrid DNA with pBR322
Hybrid DNA of 1.6NKbp fragment and pBR322
Hybrid of pBGH29 1.02Kbp and pBR322
The DNA was designated as pBGH4. The DNA of pBGH1 is
pBR322 is replicated in host E. coli.
The entire DNA was completely double-stranded. Hybrid DNApBGH1 Hind/Bgl,
Hind/Ava, Hind/Sal/Cla, Hind
/Bgl/Ava, Hind/Cla/Bgl, and then subjected to 1.2% agarose gel electrophoresis (at the same time, EcoR of λDNA was used as a size marker).
The DNA fragment size was analyzed and the results shown in Table 2 were obtained.

[Table] The fragments generated by digestion of pBGH1 with each restriction enzyme in Table 2 have a maximum of ±0.05Kbp for each DNA size.
Although there is a measurement error of about 0.1 Kbp, the result matches well with what was expected from the restriction enzyme cleavage map in Figure a, except that the Cla site 0.82 Kbp away from the Hind site is no longer cleaved by Cla. was. The Cla site 0.82Kbp away from the Hind site is
E. coli is no longer cleaved by Cla.
ATCGAT when pBGH1 replicates in HB101
This is thought to be due to methylation at position 6 N of GAT in TAGCTA. Hybrid DNApBGH1
Since pBGH1 was found to be infectious in an infectivity test using (Example i), it appears that one Cla site has disappeared, but the base sequence itself is thought to be the same as aDNA. f Hybrid DNA of Cla-digested double-stranded DNA and E. coli plasmid pBR322; In the experiment of e, double-stranded DNA and
Sterilize the double-stranded DNA that has been digested with Cla by performing almost the same procedure as e, changing the Hind digestion of pBR322 to Cla digestion (change the salt concentration during digestion to a low concentration for Cla digestion). water
17μ and pBR322 Cla/alkaline phosphatase treatment (DNA 1μg/μ) solution 1μ and the buffer for DNA binding enzyme 2μ and
Hybrids were incubated using 0.5μ of T4 DNA ligase at 14℃ for 22 hours in the same manner as in experiment e.
I got DNA. Using this hybrid DNA, E. coli HB101 competent cells were transformed in the same manner as in e. 41 transformants were obtained. Plasmid DNA was extracted from these 41 colonies using the Boiling Lysis method as in the case of e.
Make a mini preparation of this plasmid DNA
Cla decomposed. The results were 2.61Kbp, 1.33Kbp,
Three hybrid plasmids containing a 1.24 Kbp fragment were obtained. This hybrid plasmid
The DNAs were named pBGC1, pBGC2, and pBGC3.
pBGC1 is a hybrid DNA containing the entire DNA of b, pBGC2 and pBGC3 are
It was a hybrid DNA containing two Cla fragments of bDNA. hybrid
DNApBGC1 was replicated in E. coli and was completely double-stranded throughout the DNA.
Hybrid DNApBGC1 was transferred to C1a, Cla/
It was decomposed using Hind and Cla/Sal, and the field fragments were analyzed in the same manner as e. The sizes of the obtained fragments were as shown in Table 3.

[Table] The fragments generated by digestion of pBGC1 with each restriction enzyme in Table 3 include a measurement error of ±0.05 maximum ±0.1 Kbp in the size of each DNA, but the fragments shown in the restriction enzyme cleavage map in Figure b was in good agreement. The hybrid DNAs pBGH1 and pBGC1 obtained in Examples e and f are hybrid DNAs of the DNA shown in Figures a and b and pBR322, respectively.
These can also be used as vectors for plant recombination. g Separation of aDNA and bDNA from hybrid DNA pBGH1 and pBGC1 and their combined circularization; hybrid DNA pBGH1 and pBGC1 respectively
Using 20 µg of Hind20 units and 20 Cla units, set the salt concentration using the respective restriction enzyme buffers, and incubate at 37℃10 at a total of 400 µg.
Hydrolyzed for hours. Next, after electrophoresis using 0.8% agarose gel (slot width 1.5 mm, length 6 cm, depth 7 mm, gel length 13 cm) at 1 V/cm for 10 hours,
The aDNA and bDNA band parts were cut out along with the gel and the experimental book by Maniathes et al. P164-5
DNA was extracted from the gel as described, and then
DNA was purified according to the method of P166. Both were dissolved in 50μ of sterile water (approximately 0.1μg/μ of DNA). Add this linear aDNA and bDNA to 20μ each of the linear aDNA and bDNA with 5μ of the buffer for DNA ligation enzyme and sterilized water.
Add 25μ, T4-DNA ligase 0.5μ and 12℃
After reacting for 19 hours, 0.8% agarose gel electrophoresis was performed again in the same manner as above, and the DNA band with an apparent size of 3.3 Kbp was cut out from the gel, and only the DNA was extracted and purified in the same manner as described above. This DNA is circular aDNA and
It is thought that it has become bDNA. Then this
10μ of sterile water was added to each DNA, and 2μ of the water was partially degraded with Hind and Cla, respectively. For partial digestion, add 5μ of the above restriction enzyme buffer and 43μ of sterile water to a 2μ solution of DNA, then add 0.5 unit of Hind for aDNA and 0.5 unit of Cla for bDNA, and incubate at 37°C for 5 minutes. After reacting for 10 minutes and 30 minutes, each reaction product was subjected to 0.8% agarose gel electrophoresis to observe changes in size. The 3.3Kbp band gradually decreased as the reaction was carried out for 5, 10, and 30 minutes.
aDNA and bDNA linear DNA 2.57Kbp,
A band of 2.61 Kbp increased, and no other bands were observed. From this, the above circular aDNA and bDNA are both monomeric and circular, and are the DNAs in Figures a and b, respectively.
It is. This DNA is completely double-stranded throughout and can be used as a vector for plant recombination. h BGMV replicative DNA from BGMV infected plants
extraction and analysis; method of Hamilton et al. [Nucl Acids Res. 10
4902 (1982)], 40ml of
0.5MKH ₂ PO ₄ and 0.75% Na ₂ SO ₅ (PH7.0) were added and homogenized in a mortar, 1.2 ml of TritonX-100 was added, and the mixture was stirred at 4°C for 25 hours. Next, pass through double gauze, and transfer the excess portion to a No. 4 Tommy high-speed centrifuge.
Centrifuge at 9,000 rpm for 10 minutes using a rotor, remove the supernatant, and transfer this supernatant to a Betsuman ultracentrifuge type.
Centrifugation was performed at 37,000 rpm for 3.5 hours in an SW40.1 rotor. Discard the supernatant and add 1 ml to the sediment pellet.
40mM TrisHCl, 5mM acetic acid, 10mM MEDTA (PH
8.2) was added and homogenized, 20μ of 10% SDS was added, and proteins etc. were removed from the aqueous layer three times with 1 ml of water-saturated phenol and then three times with 1 ml of phenol/chloroform (4/1). Next, ether 1
Ether extraction was carried out four times to remove phenol, and to the aqueous layer were added 3P sodium acetate (100μ) and ethanol (3.5ml), and after standing at -20°C overnight, the mixture was centrifuged at high speed to obtain a DNA pellet. Add 500μ of sterile water to this DNA pellet (DNA concentration: 0.6μ).
g/μ) The 100μ was subjected to 0.8% agarose gel electrophoresis using the same method as for 1g.
DNA corresponding to 1.5 Kbp of the band that hybridizes with the 32p labeled DNA probe of BGMV,
DNA equivalent to 3.1Kbp, DNA equivalent to 5.2Kbp
The 9.8 Kbp portion of DNA was extracted from the gel and purified in the same manner as in g.
(The portion equivalent to 5.2Kbp and the portion equivalent to 9.8Kbp are λ−
Using DNA EcoR/Hind size markers, a corresponding portion of each gel was cut out). These four types of DNA are respectively Hind, Cla, and Sal.
The decomposition pattern was analyzed by performing agarose gel electrophoresis. 5.2Kbp equivalent portion,
For the DNA corresponding to 9.8 Kbp, the DNA after agarose gel electrophoresis was transferred to a nitrocellulose filter, and the 32p-labeled BGMV was transferred to a nitrocellulose filter.
Perform DNA-DNA hybridization and autoradiography using DNA.
The decomposition pattern of was analyzed. The decomposition pattern is 2.58, 1.60, 1.00Kbp,
2.57, 1.33, 1.24Kbp, Sal by Cla decomposition
Degradation patterns showed degradation patterns of 1.90, 1.54, 1.03, and 0.71 Kbp. This is the same pattern of digestion by restriction enzymes a and b,
That is, this replicative DNA is DNA-a or DNA-
b, and can be used as a vector for plant recombination. i Hybrid of replicative DNA and pBR322
Preparation of DNA: DNA (9 μg DNA/10 μ) extracted and purified from agarose gel of a band portion with an apparent size of 1.5 Kbp among the replicated DNA of h, was added with 4 μ of the Cla restriction enzyme buffer and sterile water.
24μ of Cla and 3μ of Cla were added and hydrolyzed at 37°C for 2 hours, 60μ of sterilized water was added thereto, protein was removed twice with 60μ of water-saturated phenol, and then phenol was extracted from the aqueous layer four times with 130μ of ether. Next, add 7μ of 3M sodium acetate (PH4.8) and 300μ of ethanol at −80℃.
After standing for 30 minutes, the mixture was centrifuged at high speed to obtain a precipitate. cold
The precipitate was washed with 500μ of 70% ethanol, centrifuged again, and the supernatant was discarded, and the precipitate was dried at 2 mmHg for 3 minutes. This precipitate is dissolved in 20μ of sterile water, and this is used as a Cla degradation product of replicative DNA.
Add f to 4μ of this Cla decomposition product (approx. 2μg of DNA).
The same pBR322 Cla digestion/alkaline phosphatase treatment solution used in
) 6μ, buffer for the DNA-binding enzyme 2μ
and T4 DNA ligase 0.5μ, sterile water 7.5μ
In addition, the reaction was carried out at 14℃ for 12 hours, and then treated at 65℃ for 5 minutes.
Competent cells were transformed and approximately 500 transformants were obtained. L- in which colonies of transformants have arisen
Place a nitrocellulose filter (BA85 manufactured by Schleicher and Schiill; same as above) on the Agar plate, then remove the nitrocellulose filter and soak it in 0.5M sodium hydroxide and 1.5M saline for 1 minute. , and then immersed in 1M TrsHCl (PH7.0) solution. This nitrocellulose filter was heated to 80°C for 2 hours to 1mm
The pressure was reduced to Hg and heat treated. Add this heat-treated filter to prehybridization solution [0.9M]
NaCl, 0.09M sodium citrate, 0.02% polyvinylpyrrolidone (Wako Pure Chemical Industries, Ltd. Plasdone NP)
K-30) 0.02% Ficoll (Ficoll 400, manufactured by Pharmacia Fine Chemicals) 0.02% bovine serum albumin aqueous solution], pretreated at 63°C for 30 minutes, then removed the prehybridization solution and pre-hybridized anew. A hybridization solution containing 10% SDS and 40 μg/ml of yeast tRNA was added to the dissolution solution, and 200 x 10 ⁴ cpm of the BGMV 32p probe described below was added to the solution. Add cellulose filter and DNA-DNA at 63℃ for 24 hours.
Hybridization was performed. Next, take out this filter, add a large amount of 0.45M NaCl,
The filter was washed by gentle shaking in 0.045M sodium citrate at 55°C for 20 minutes. This washing was carried out three times, and then after drying, using this filter,
Autoradiography was performed using the same method as 471. Approximately 360 colonies were observed that hybridized with 32p-labeled BGMV DNA. Preparation of BGMV 32p probe: In experiment c, 0.8mM deoxycytidine triphosphate was used instead of 10μ to 0μ. Other procedures were carried out in the same manner as in c, and No.
The first 32p peak in the 6th to 11th floor fractions (total 800
These fractions were collected (tctal 900μ), heated at 95°C for 5 minutes, and then rapidly cooled to ⁰ °C. This liquid is 32p labeled BGMV.
Use as a DNA probe. Plasmid DNA was mini-prepared from 30 of the 360 colonies by the Boiling Lysis method described above, and the plasmid DNA was digested with Cla. As a result, a hybrid plasmid containing a 2.61 Kbp fragment was obtained from 7 colonies. One of these hybrid DNAs was named pBGRFC1. Cla/Hind pBGRFC1
, Cla, and Sol showed the same DNA fragment as in Table 3 of f. j Hybrid of replicative DNA and pBR322
Preparation of DNA; DNA extracted and purified from the band portion of the replicative DNA of h with a size equivalent to the apparent size of 3.1 Kbp.
is decomposed by Hinp, and this is done similarly to e.
Hybrid DNA was obtained with pBR322 treated with Hind/alkaline horsphatase, and E. coli was further transformed. Plasmids were extracted and analyzed from 30 of the 161 transformants obtained in the same manner as e. Hybrid DNA containing Hind fragments of 2.58Kbp, 1.59Kbp, and 0.99Kbp at the Hind site of pBR322, respectively pBGRFH1,
pBGRFH2 and pBGRFH3 were obtained. pBGRFH1 is Hind/Bgl, Hind/
Ava, Hind/Cla/Sal decomposition e
DNA fragments similar to those in Table 2 were generated.
From the results of i and j, this replicative DNA is shown in the figure.
It was confirmed that it contained aDNA and bDNA. Using the method of this experiment, aDNA,
Each bDNA can be isolated and propagated. k Infectivity by cloned DNA; hybrid DNA pBGH1 (2.3μ) obtained in e.
g/μ) 25μ to 2mMMgCl ₂ 10μ, above
Hind restriction enzyme buffer 13μ, Hind
Add 5μ and 77μ of sterile water and treat at 37℃ for 7 hours.
After further treatment at 65°C for 5 minutes, NaCl and sodium citrate were added until the concentrations were 0.15M and 0.015M, respectively. This solution was used as a cloning aDNA solution. Hybrid DNA pBGC6 obtained in f.
(0.68μg/μ) 10μ of _2mMMgCl2 in 100μ,
Buffer 13μ for Cla restriction enzyme, Cla
Add 5μ of sterilized water and 2μ of sterilized water and treat at 37℃ for 7 hours.
After further treatment at 65°C for 5 minutes, NaCl and sodium citrate were added until the concentrations were 0.15M and 0.015M, respectively. This solution was used as a cloning bDNA solution. Cloning aDNA solution and
Mix 80μ of each bDNA solution and store the mixture at 30°C for 14 hours during the day and at 27°C for 10 hours at night.
In a Biotron (Koitotron manufactured by Koito Manufacturing Co., Ltd.) set at a humidity of 80% or higher, the surfaces of young leaves of Topkurotsupin bean, 6 days after germination, were inoculated by rubbing together with Celite using sterile gloves (mixed solution 40μ/1 Individual top crop beans). A total of 4 individuals were inoculated. Thereafter, the conditions in the biotron were maintained as described above, and the onset of bean golden mosaic disease was observed on the true leaves of top-cropped kidney beans. Ten days after inoculation, bean golden mosaic disease was clearly observed in all four individuals. From the above, cloning aDNA and
When bDNA is cloned, a special base sequence is methylated, for example N at position 6 of A of GATC, and specific restriction sites (for example, Cla site 1 of the restriction enzyme cleavage map in Figures a and b) are
Even though it appears that the DNA has disappeared and is different, the base sequence itself is the same.
Since it exhibits the same infectivity as DNA, it is recognized to have the same base sequence as viral DNA. k Infectivity by cloned DNA; hybrid DNA pMYB4 obtained in e.
(0.435μg/μ) 2mMgCl ₂ 10μ to 460μ,
55μ buffer for the BamH restriction enzyme,
After adding 10μ of BamH and 15μ of sterile water and enzymatic treatment at 37℃ for 4 hours, further treatment at 65℃ for 5 minutes.
55μ of 1.5M NaCl and 0.15M sodium citrate aqueous solution was added. Cloning this liquid
It was made into aDNA solution. Hybrid obtained by f
DNApMYH3 (0.87μg/μ) to 231μ
10μ of 2mMMgCl ₂ , 30μ of the Hind restriction enzyme buffer, 10μ of Hind, and 19μ of sterilized water were added, and the mixture was treated with enzymes at 37°C for 4 hours. After further treatment at 65°C for 5 minutes, 30μ of 1.5M NaCl and 0.15M sodium citrate aqueous solution were added. This solution was used as a cloning bDNA solution. Cloning aDNA solution 330μ and
Mix 160μ of bDNA solution, and add this mixture to
30℃ for 14 hours in daytime condition, 10 hours in nighttime condition 27
In a Biotron (Koitoron manufactured by Koito Seisakusho Co., Ltd.) set at ℃ and humidity of 80% or higher, inoculation was inoculated by rubbing it with Celite on the surface of young leaves of Topkurotsupin bean on the 6th day after germination using sterile gloves. / 1 individual top cropped green bean). A total of 10 individuals were inoculated. Thereafter, the conditions in the biotron were maintained in the same manner as described above, and the appearance of yellow mosaic lesions was observed on the true leaves of the top crop kidney bean. On the 18th day after inoculation, yellow mosaic lesions were clearly observed in 9 out of 10 individuals. From the above, the cloned aDNA and bDNA obtained in the present invention have a special base sequence, for example, A6 of GATC, by cloning.
Even if the N at position is methylated, etc., the virus
Shows the same infectivity as DNA. From this, it can be considered that they have substantially the same base sequence.

[Brief explanation of drawings]

Figures a and b are DNA according to the present invention, respectively.
This shows a restriction enzyme cleavage map of .

Claims (1)

[Claims] 1. Single strand of bean golden mosaic virus
The DNA is at least partially double-stranded, and is one of the DNAs represented in the genetic map below.
~Trimeric DNA. [Here, the base sequence of one strand of the double strands of P ₁ to _{P 7} is as follows. P ₁ (0.0/2.57): 5′−AAGCTT−3′ P ₂ (0.82): 5′−ATCGAT−3′ P ₃ (0.97): 5′−AGATCT−3′ P ₄ (1.18): 5′− GTCGAC−3′ P ₅ (1.38) :5′−CPyCGPuG−3′ P ₆ (2.08) :5′−ATCGAT−3′ P ₇ (2.21) :5′−GTCGAC−3′] 2 In other organisms Claim 1 characterized in that at least a part of it is methylated.
DNA described in section. 3 Claim 2 in which the other organism is E. coli
DNA described in section. 4. The DNA according to claim 1, which has been double-stranded in vitro. 5. The DNA according to claim 1, which has been double-stranded in vivo. 6. The DNA according to claim 1, which is a monomer. 7 Bean golden mosaic virus single strand
Hybrid DNA is DNA that is at least partially double-stranded and includes DNA represented by the genetic map below. [Here, the base sequence of one strand of the double strands of P ₁ to _{P 7} is as follows. P ₁ (0.0/2.57): 5′−AAGCTT−3′ P ₂ (0.82): 5′−ATCGAT−3′ P ₃ (0.97): 5′−AGATCT−3′ P ₄ (1.18): 5′− GTCGAC−3′ P ₅ (1.38) :5′−CPyCGPuG−3′ P ₆ (2.08) :5′−ATCGAT−3′ P ₇ (2.21) :5′−GTCGAC−3′] 8 In other organisms 8. The hybrid DNA according to claim 7, wherein at least a portion of the hybrid DNA is methylated. 9 Claim 8 in which the other organism is E. coli
Hybrid DNA as described in Section. 10. The hybrid DNA according to claim 7, which has been double-stranded in vitro.